The present invention provides novel compositions of matter and novel methods of using these compositions of matter. In particular, the present invention provides novel recombinant DNA (rDNA) compounds and methods for incorporating these novel compounds into the expressed genetic material of cells and cell lines, whereby novel cells and cell lines are thereby produced and comprise yet another element of the invention herein. Most particularly, the present invention relates to the incorporation of recombinant DNA compounds into the expressed genetic material of higher eukoryotic cells for expression of pre-selected polypeptides.
Methods of synthesis of recombinant DNA compounds are well known in the art. Moreover, these techniques have been extensively cataloged. Refer, for example, to T. Maniatis, et al., "Molecular Cloning: A Laboratory Manual," Cold Spring Harbor Laboratory Cold Spring Harbor, N.Y. (1982). Also for a general discussion of recombinant DNA compounds and their synthesis and use in molecular biology, refer to J.D. Watson, et al., "Recombinant DNA: A Short Course," Scientific American Books, New York, N.Y. (1983), and the extensive bibliography of references cited therein.
DNA compounds in accordance with the present invention are those biologically useful polymers of deoxyribonucleic acid linked together by 5'-3' phosphodiester bonds between the sugar (deoxyribose) and phosphate groups. The DNA polymers are those composed of repeating nucleotide units and, as disclosed herein are capable of existing in the double helix configuration wherein the paired guanine/cytosine (G/C) and adenine/thymine (A/T) bases form hydrogen bonds stabilizing the double helix.
DNA compounds are known to exist in all or essentially all living cells. These DNA compounds when incorporated into the expressed genetic material of cells, e.g., properly incorporated as elements of genes, plasmids, and chromosomes, are capable of transcribing into messenger ribonucleic acid (mRNA) the ordered base pair sequences of the DNA. The process of transcription into RNA is part of the process which enables polypeptide and/or protein synthesis within living cells to be accomplished. The techniques of molecular biology now permit recombinant DNA compounds to be incorporated into the expressed genetic material of living cells for the purpose of transcribing into messenger RNA coding for pre-selected polypeptides other than those which a cell might be capable of synthesizing prior to the incorporation or permit the cell to synthesize its natively-produced polypeptides at much higher expression levels.
Molecular biology research often begins by the isolation and characterization of DNA compounds from native sources. For example, a DNA compound has been isolated from the adult bovine pituitary which transcribes into messenger RNA the base pair coding which directs the synthesis of bovine growth hormone or bGH. The isolation and characterization of this DNA compound is described in European published patent application 0112012, published June 27, 1984. Because this DNA compound was derived from the bovine pituitary gene responsible for bGH expression, such a compound is commonly referred to as genomic DNA or gDNA.
In general, messenger RNA derived from this gDNA by transcription can then be copied into DNA utilizing reverse transcriptase enzymes. This copying of an RNA base sequence onto DNA yields a DNA compound commonly referred to as copy DNA or cDNA. For mammalian genes, the sequence of cDNA often differs from the sequence of gDNA coding for the same polypeptide or protein in that the gDNA often contains introns or non-polypeptide coding sequences in addition to the codons. Refer, for example, to the known bGH gDNA containing four such introns between the 5' and 3 ends. Those ordered gDNA sequences with ordered counterparts on this mRNA transcribed there. from are, accordingly, often referred to as "exons".
Genes capable of expression contain flanking sequences beyond the exons (the portions of DNA that are transcribed onto messenger RNA) which do not code for messenger RNA but are nonetheless essential to the transcription process. Upstream (above the 5' end) from the site where messenger RNA transcription begins is a DNA sequence responsible for promotion or initiation of transcription which is commonly referred to as the "promoter"In most eukoryotic cells, a region of DNA downstream (below the 3' end) from the last exon is responsible for the addition to messenger RNA of a long sequence of adenine-containing nucleotides which are added after transcription is completed. This region thus signals the addition of a "poly-adenine tail" also known as the polyadenylation of the messenger RNA.
For some proteins certain of the requirements for this poly. adenylation signal are known. See, for example, R.P. Woychik, et al., "Requirement for the 3' Flanking Region of the Bovine Growth Hormone Gene for Accurate Polyadenylation", Proc. Natl. Acad. Sci. USA 81:3944-3948 (July 1984) Also, the nucleotide sequence AATAAA characterizes the polyadenylation signal at a location 11 to 30 nucleotides upstream (towards the 5' end) from the 3' end of the gene. Refer to D.R. Higgs, et al., Nature 306:398-400 (Nov. 24, 1983) and references cited therein.
The present specification describes a method for utilizing a bGH polyadenylation signal in conjunction with DNA especially cDNA. coding for a pre-selected polypeptide other than bGH as a means of obtaining expression of the pre-selected polypeptide in a higher eukaryatic cell.